Project Summary The M2 protein of influenza A and B viruses (AM2 and BM2) forms an acid-activated proton (H+) channel for virus entry and mediates membrane scission in a cholesterol-dependent fashion for virus budding. AM2 is inhibited by the amantadine class of antiviral drugs until the recent emergence of drug-resistant M2 mutants among circulating flu viruses, and no antiviral drugs are yet available against BM2. Thus, structural and mechanistic studies of M2 are important for designing new M2 inhibitors to curb seasonal and pandemic flu. Due to its modular nature and its small size, the M2 protein also serves as a model system for understanding the structural principles governing H+ transport in ion channels and the mechanism of membrane- curvature induction by proteins. So far, the structural basis for how M2 prevents reverse H+ current from the C-terminus to the N-terminus is not yet known. How the N-terminal ectodomain and the C-terminal cytoplasmic tail modulate drug-sensitive H+ conduction through the transmembrane (TM) pore and induce membrane curvature is poorly understood. Structural information about M2 interaction with cholesterol is scarce. Finally, the structure of influenza BM2 in lipid bilayers has not been investigated, and mechanistic information about how BM2 conducts protons is sparse. We propose to employ solid-state NMR spectroscopy to answer these structural and mechanistic questions about influenza AM2 and BM2 in phospholipid bilayers. In Aim 1, we will investigate the H+ conduction dynamics and drug binding equilibrium of fully functional AM2 containing the ectodomain and the cytoplasmic tail. 2D correlation experiments that detect both TM and extra-membrane residues and 2H NMR experiments that probes drug orientation and dynamics will be performed. In Aim 2, we will investigate the structure, dynamics and H+ conduction mechanism of BM2. The sidechain conformation and inter-residue contacts of His and Trp in the conserved HxxxW motif will be measured, and hydration of the channel residues will be investigated using 1H-13C correlation experiments. In Aim 3, we will study gating-deficient mutants of AM2 to understand how Trp41 and Asp44 ensure unidirectional H+ flow from the N-terminus to the C-terminus. Sidechain conformation, dynamics, and inter-residue distances among the key functional residues will be measured. In Aim 4, we will probe M2-cholesterol interactions by measuring cholesterol orientation and dynamics, cholesterol-induced chemical shift changes, and intermolecular distances to constrain the putative M2-cholesterol complex.